A unique proteolytic fragment of human fibrinogen containing the A alpha COOH-terminal domain of the native molecule.

The COOH-terminal portion of the A alpha chain of human fibrinogen is highly susceptible to proteolytic degradation. This property has prevented isolation of the COOH-terminal domain of fibrinogen for the direct investigation of its functional characteristics. Human fibrinogen was degraded with hementin, a fibrinogen-olytic protease from the posterior salivary glands of the leech, Haementeria ghilianii. Two initial fragments, Yhem1 and Dhem1, produced by cleavage through the three polypeptide chains in the connector region, were characterized and shown to retain the entire A alpha COOH-terminal domain. Late cleavages by hementin occurred in the A alpha chain COOH-terminal region to produce fragments Yhem and Dhem with shorter A alpha chain remnants. Fragments Dhem were isolated from an intermediate hementin digest of fibrinogen using anion-exchange chromatography. Fragment Dhem1 was separated further from Dhem fragments with shorter alpha chain remnants by affinity chromatography on immobilized plasma fibronectin. Fragment Dhem1 represents a unique proteolytic fragment of fibrinogen containing an intact A alpha chain COOH-terminal region. NH2-terminal sequence analysis of isolated chains from fragment Dhem1 located hementin cleavage sites in the connector region to A alpha Asn102-Asn103, B beta Lys130-Gln131, and gamma Pro76-Asn77. The specific interaction of fragment Dhem1 with immobilized fibronectin indicated that the binding site probably was located within the COOH-terminal 111 amino acids of the A alpha chain. The overall pattern of fibrinogen cleavage by hementin is similar to that of plasmin, yet hementin cleaves preferably in the coiled-coil connector, sparing the A alpha COOH-terminal domain.

The specific interaction of fragment Dhem 1 with immobilized fibronectin indicated that the binding site probably was located within the COOHterminal 111 amino acids of the Aa chain. The overall pattern of fibrinogen cleavage by hementin is similar to that of plasmin, yet hementin cleaves preferably in the coiled-coil connector, sparing the Aa COOH-terminal domain.
Fibrinogen, a tbrombin-coagulable plasma glycoprotein, consists of three pairs of disulfide-bonded chains called ALY, BP, and y. The polypeptide chains are folded into four structural domains: the D, E, connector, and the COOH-terminal region of the Acu chain.
Plasmin attacks the ALY chain COOH termini of fibrinogen first (l-4), resulting in the production of fragment X (250 kDa) (5,9), that is heterogeneous (2,6) as a result of multiple cuts in the Aa chains and cleavages which release the NH*terminal, fibrinopeptide B-containing peptides from the B@  (7,8). Cleavage in one of the two connector regions of fragment X results in the production of fragments Y (155 kDa) and D (100 kDa). Cleavage in the connector region of fragment Y results in the production of a second fragment D and a single fragment E (50 kDa) (10-12). Fragments D and E, which are terminal digestion products (5, 9), represent the major globular domains in fibrinogen (10-12). Fibrinogenolytic enzymes have been discovered in leukocytes (13) and platelets (14) and have been implicated in an alternative in uiuo pathway for fibrinolysis (15). Although leukocyte and platelet proteases give different cleavage patterns than plasmin, the blood cell enzymes attack the Aa chain first (16)(17)(18)(19). Fibrinogenolytic proteases isolated from some snake venoms also cleave fibrinogen in the Aa chains preferably before attacking the other chains (20)(21)(22)(23)(24). The Aa chains are also cleaved first by trypsin (6).
The giant South American leech Haementeriaghilianii contains a fibrinogen-degrading protease, hementin, which is present in the posterior and anterior salivary glands (25). The present study aimed at elucidating the pattern of fibrinogen degradation by hementin from the posterior salivary glands of the leech. Structural characterization of fibrinogen degradation fragments was performed and a unique fragment called D he,,, 1, which retained an intact COOH terminus of the native molecule, was purified. The structure of fragment Dhem 1 was characterized, and its binding to plasma fibronectin was demonstrated. 13669 allowed to stand for 15 min at room temperature. The mixture was then allowed to cool to 4 "C before homogenization. Homogenization was with a Tri-R-stir-r Model K41 homogenizer (Tri-R instruments, Rockville Center, NY). The procedure was carried out in four 10-s bursts. In between homogenizations, the preparation was allowed to cool to 4 "C. The homogenized leech glands were centrifuged at 12,000 x g in a fixed angle rotor using a Sorvall RC-2 centrifuge. The supernatant was taken and stored at 4 "C while the leech gland pellet was re-extracted. The combined supernatants were then centrifuged at 100,000 x g, aliquoted, and stored at -20 "C. Extracts prepared in this manner were stable indefinitely at -20 "C.
Digestion of Band I Fibrinogen with Hementin-Band I fibrinogen, 2 mg/ml final concentration, was digested with 8 rg/ml of gland extract containing hementin. Digests were made in either 150 mM HEPES, 10 mM CaCl*, pH 7.8 buffer, or 0.05 M ammonium bicarbonate, 10 mM CaC12, pH 7.8 buffer at 37 "C. Digestion was terminated by adding EDTA to 10 mM final concentration, and the extent of degradation was determined by SDS-PAGE (26).

Immunological
Characterization of Fibrinogen Fragments-Polyclonal antisera against fibrinogen, fragment D, fragment E, and the Aa chain were obtained in rabbits. The antisera were absorbed with normal human serum. Antisera to fragment D were also absorbed with purified fragment E, antisera to fragment E were absorbed with purified fragment D. Anti-D and anti-E antisera did not cross-react as judged by immunoelectrophoresis (27). Monoclonal antisera against epitopes on the Aa chain COOH terminus were obtained and characterized as previously described (28). The immunoblot procedure of Towbin et al. (29) was used to characterize fibrinogen fragments by reaction with antibodies against specific fibrinogen domains.

Characterization
of the Polypeptide Chain Composition of Fibrinogen Fragments by Two-dimensional SDS-PAGE-In the first dimension, the fragments were separated by SDS-PAGE on a 3.5-5.0% polyacrylamide slab gel gradient under nonreducing conditions. A 1.0 x 15.9-cm lane with separated fragments was cut out and incubated in 10 mM DTT at 37 "C for 30 min. The reduced gel was placed at a right angle onto a 9.0% polyacrylamide slab gel and sealed with 1.0% agarose. Electrophoresis was then carried out at 20 mA until the bromphenol blue dye front reached the bottom of the gel. Protein spots were visualized by Coomassie Brilliant Blue R-250 staining. A similar gel was also electroblotted onto nitrocellulose and incubated with antibodies to the Acu chain of fibrinogen. Bands were visualized with Protein A Gold (Bio-Rad).

Separation of Fibrinogen
Fragments by Anion-exchange Chromatography-Anion-exchange chromatography of a hementin digest of fibrinogen was carried out on a Mono Q HR 5/5 column using the Pharmacia FPLC system with an automated gradient controller (Pharmacia). Samples were prepared for chromatography by extensive dialysis against 0.05 M ammonium bicarbonate, 1 mM EDTA, pH 8.0, followed by filtration of the sample through a 0.22 pm Millipore syringe filter (Millipore Corp., Bedford, MA). The sample was applied to the column in 0.05 M ammonium bicarbonate, pH 8.0, and washed to remove any unbound protein. Bound protein was eluted with a linear NaCl gradient in ammonium bicarbonate buffer, pH 8.0: (a) from 0 to 0.2 M in 40 ml, (b) from 0.2 to 0.5 M in 3 ml; and (c) 3-ml isocratic elution at 0.5 M NaCl. Protein was eluted at 1.0 ml/min and collected in 0.65ml fractions. Six mg of protein were separated per run.

Purification and Immobilization of Plasma
Fibronectin-Plasma fibronectin was purified from fresh human plasma using affinity chromatography on immobilized gelatin by a modification of the procedure by Engvall and Ruoslahti (30). Sixty mg of purified fibronectin in 0.1 M HEPES, pH 6.5 buffer, containing 0.01% Tween 20 was coupled to 4 ml of Affi-Gel 15 (Bio-Rad) according to the manufacturer's instructions. The coupling efficiency was >95%. preparation containing fragments Dhem was obtained after chromatography of a hementin digest on Mono Q followed by chromatography on immobilized anti-E antibodies. One mg of the preparation was applied to a column containing 200 mg of immobilized fibronectin. The sample was applied in phosphate-buffered saline, pH 7.4, and washed with the same buffer at a flow rate of 1.0 ml/min. One-ml fractions were collected. The elution buffer was then switched to 0.01 M phosphate, 1.0 M NaCl, pH 7.4, and the elution procedure repeated. Fragments Dh em , were then eluted from the column using a linear gradient composed of 35 ml of 0.01 M phosphate, 1.0 M NaCl, pH 7.4, as the start buffer and 35 ml of 0.01 M phosphate, 1.0 M NaCl, 4.0 M urea, pH 7.4, as the gradient buffer. Fragment Dhem, was eluted at a urea concentration of 1.0 M.

Reduction and Carboxymethylation of Fragment
Dhrm ,-The reduction and carboxymethylation of polypeptide chains derived from purified fragment Dheml was done by a modification of the procedure of Bewley et al. (31). One mg of lyophilized fragment Dhem , was dissolved in 1.0 ml of 0.2 M Tris-HCl, 6.0 M guanidine HCl buffer. DTT was added to the fragment Dhem , solution at a 1500-fold molar excess. Reduction was allowed to proceed at 90 "C for 30 min. The reaction mixture was cooled to room temperature using ice water. Iodoacetic acid was added in a 3.0-fold molar excess over DTT. Incubation took place in the dark at room temperature for 20 min and on ice for 30 min. Unreacted DTT and iodoacetic acid were removed by gel filtration. The carboxymethylated chains of fragment Dhem , were analyzed by 5.0-10.0% SDS-PAGE under nonreducing conditions.

Separation of Reduced, Carboxymethylated Fragment
Dheml Chains by Reverse-phase HPLC-Four hundred gg of lyophilized CM-DI,,, 1 chains were dissolved in a 1:l mixture (v/v) of 0.1% trifluoroacetic acid, 10% acetic acid, to 1.0 mg/ml final concentration. Chromatography was carried out on a Vydac C, reverse-phase HPLC column, 25 cm X 4.6 mm inner diameter (Vydac, Hesperia, CA), using a Waters HPLC system with a dual pump and an automated gradient controller (Waters Assoc, Inc., Milford, MA). The start solution was A timed digest of fibrinogen (Fg) was analyzed by nonreduced SDS-PAGE on 3.5-5.0% polyacrylamide gradients. Fragments produced in a 24-h digest are characterized by assigned name and molecular weight, calculated from protein standards. The nomenclature for fibrinogen fragments produced by hementin has been derived from that for plasmic fragments. The average sizes for plasmic fragments determined by many investigators using many methods are listed in the left-hand column. The sizes calculated for fibrinogen fragments produced by hementin are listed in the center column. The differences in mass among the fibrinogen fragments produced by hementin are in the right-hand column. Comparison of mass between fibrinogen fragments was used as a basis for evaluating structures of hementin-generated fibrinogen fragments.
Plasmic Fibrinogen (Fg) was first digested for 24 h with hementin to produce an intermediate digest containing fragments Yhem, Dhem, and Ehem. The sample was split into two aliquots. One sample was incubated at 37 "C for 30 min in the presence of 10 mM EDTA (first lane), the other, in the presence of 0.5 CTA units/ ml plasmin (second lane). A mixture of plasmic fragments X, Y, D, and E, is shown in the third lane. 0.1% trifluoroacetic acid. The gradient was made with 100% acetonitrile, 0.1% trifluoroacetic acid. CM-Dh., , chains were eluted using a 40-ml linear gradient from 30-60% acetonitrile. The flow rate was 1.0 ml/min, and 0.5-ml fractions were collected. The column was washed free of all other bound material with 100% acetonitrile and re-equilibrated with 0.1% trifluoroacetic acid.

RESULTS
Unique Fibrinogen Fragments Produced by Hementin--The pattern of fibrinogen digestion by hementin was determined by characterizing the structures of fragments formed in a timed digest (Fig. 1). The molecular weight of each fragment formed, calculated relative to standards on SDS-PAGE, was used for evaluating fragment structures.
Early cleavage of fibrinogen produced two fragments of 204 and 140 kDa simultaneously.
The two fragments persisted as the major products for the first 8 h of digestion (Fig. 1). Late cleavages produced additional lower molecular weight fragments after 24 h (Fig. 1) An intermediate hementin digest was separated by SDS-PAGE and electroblotted onto nitrocellulose. Three separate samples of the same digest were developed with polyclonal antibodies to either the native fibrinogen molecule, plasmic fragment D, or plasmic fragment E. two fragments was calculated at 344 kDa, a value comparable to the mass of the intact fibrinogen molecule. Furthermore, release of smaller peptide material was not detected by SDS-PAGE (data not shown). Using this information in conjunction with the documented model of fibrinogen structure and its degradation by plasmin, we postulated that hementin cleaved first through the three polypeptide chains in the connector region to split the fibrinogen molecule asymmetrically. Since plasmin also cleaves through the connector region of fibrinogen, producing the asymmetric fragment Y, followed by fragments D and E (5,9), nomenclature for describing the fragments produced by hementin was derived from that developed previously for plasmic fragments. Plasmin cleaves the Aa! chains first forming fragment X of 250 kDa (Table I) (5,9). Hementin cleavage did not result in the formation of a fragment of comparable size. Therefore, the hypothesis that the early fragments Yhem 1 and Dhem 1 retain their Aa chain COOH termini was investigated.
The hypothesis is consistent with the difference in mass between fragments produced by plasmin and hementin ( A, fibrinogen fragments formed in an intermediate hementin digest were separated in the first dimension on nonreduced 3.5-5.0% SDS-PAGE. The lane containing separated fragments was reduced in 10 mM DTT and attached to a 9.0% polyacrylamide gel at 90 ' relative to the original separation. Reduced polypeptide chains of each fragment, separated in the second dimension, were visualized by Coomassie Blue staining. Fg std., reduced chains of standard fibrinogen were separated in the second dimension only. Polypeptide chain migrations are compared with reduced native fibrinogen chains. B, to demonstrate o( chain degradation more directly, an identical gel to that shown in A was electroblotted onto nitrocellulose followed bv development with polyclonal antibodies against the entire Ao! chain. I). The difference in mass between fragments Yhem 1 and Y is 49 kDa, corresponding to the mass of the Aa chain COOHterminal peptide and the BP NH2-terminal peptide released from fibrinogen by plasmin. The mass difference of 40 kDa calculated for fragments Dhem 1 and D1 is the mass of the Aa chain COOH-terminal peptide.
The structure of fragment Ehem was evaluated according to its calculated size (62.5 kDa) and its timed appearance in the fibrinogen digest (Fig. 1). Fragment Ehem first appeared at 4 h, the band intensity increasing with time. The progressive increase in the amount of fragment Ehem paralleled the decrease in fragments Yhem. Longer digestion resulted in the retention of a constant amount of fragment Ehem (data not shown). Therefore, it was concluded that fragment Ehe,,, is derived from fragments Yhem by cleavage through the three polypeptide chains in the connector region in a manner similar to the generation of fragment E from fragment Y by plasmin. Y hem 3 (9 kDa), Dhem 2 and Dhem 3 (9 kDa). The data suggested a precursor-product relationship between fragments Yhe,,, 1 and smaller fragments Yhem, as well as Dhem 1 and smaller fragments Dhem. The similarity in size of peptides released to form both of the smaller fragments Yhe,,, and Dhem indicated a cleavage in the COOH terminus of each fragment because the two COOH termini are identical. Furthermore, the total mass of material released upon conversion of fragment Dhem 1 to fragment Dhem 4 (38 kDa) is consistent with cleavage in the cr chain as judged from the mass available for cleavage from each chain on the COOH-terminal side of the disulfide ring. A direct comparison between the cleavage sites of hementin versus plasmin was made by comparing fibrinogen fragments formed by (1) cleavage with hementin, (2) cleavage with hementin followed by plasmin, and (3) Fig. 2) indicating that some plasmic cleavage sites were still available in fibrinogen fragments produced by hementin. All three Yhem fragments were cleaved to a single plasmic fragment Y, and the four Dhem fragments were cleaved to plasmic fragment Dr, most likely by cleavage of the Aa chain COOH-terminal peptide. The results are consistent with the idea that fragments Yhe,,, and Dhem are similar to their plasmic counterparts with the addition of varying extensions of the Aa chain. The cleavage patterns also indicate that plasmin and hementin have cleavage sites in the connector region which are in close proximity.
On the basis of the parallel structures inferred for hementin and plasmic fragments, an immunologic approach to the characterization of fragments Yhem, Dhem, and Ehem was taken using antibodies specific for plasmic fragments D and E. When a control immunoblot of an intermediate hementin digest of fibrinogen was developed with antiserum against fibrinogen, a positive reaction occurred with all components of the digest (Fig. 3, first lane). However, when identical immunoblots were developed with either anti-fragment D or anti-fragment E antibodies, fragments Yhem reacted positively under both conditions, fragments Dhem reacted only with anti-D antibodies, and fragment Ehem gave a positive reaction only with anti-E antibodies, as predicted (Fig. 3, second and third lanes).
The polypeptide compositions of fibrinogen fragments were characterized by two-dimensional nonreduced and reduced SDS-PAGE. The fragments were first separated by SDS-PAGE under nonreducing conditions. After reduction of disulfide bonds, chains were separated in the second dimension (Fig. 4A). The chains of fragment Yhe,,, 1 comigrated with the chains of the reduced fibrinogen standard, demonstrating that fragment Yhe,,, , contained an intact half of the fibrinogen molecule. The two-dimensional electrophoretic pattern also demonstrated Aa chain cleavage to produce both fragments Yhem 2 and Yhe,,, 3. The BP and y chains remained intact. The (Y chain remnant in fragment Dheml had a calculated mass of 59 kDa, larger than the intact BP chain, signifying the retention of the entire (Y chain COOH-terminal region. The sizes Identical samples of an electroblotted digest were developed with monoclonal antibodies to the Aa chain COOH terminus. In the first lane a mixture of monoclonal antibodies to epitopes spanning the entire COOH-terminal portion was used. In the second lane, monoclonal antibody (MoAb) 9E9 was used. The epitope of mAb 9E9 has been mapped to ALU"~~~"~~ (28). of the /3 and y chain remnants in fragments Dhem 1-3 remained the same. The Aa and 01 chain remnants in fragments Yhem and Dhem shown in Fig. 4A displayed a diagonal pattern indicative of cleavage. The pattern formed because molecular weight differences between fragments were solely due to (2 chain cleavage. To directly demonstrate that the late cleavages were made only in the o( chain, a gel identical to that illustrated in Fig. 4A was electroblotted onto nitrocellulose and developed with antibodies to the Aol chain. Fig. 4B shows the resulting diagonal pattern of o( chain cleavage. Only the ALX chains from fragments Yhem ,, Yhem *, Dhem 1, and Dhem 2 appeared on the immunoblot, as determined by comparing the distances of the diagonal patterns between the Coomassie Blue-stained gel and the immunoblot.
The next experiment tested the hypothesis that late cleavages in the Aa chain and (Y chain remnant of fragments Yhem I and Dhem 1, respectively, took place in the COOHterminal portion. A mixture of monoclonal antibodies to epitopes within the (Y chain COOH-terminal region, or a single monoclonal antibody (mAb 9E9) whose epitope occurs between amino acids 509 and 583 (28), were used to develop similar electroblots of fibrinogen digests. Fig. 5 shows the reaction of fibrinogen, fragments Yhem ,-3, and Dhem 1-3 with the antibody mixture (first lane). However, only fibrinogen, The elution profile and SDS-PAGE analysis of selected fractions from a separation of an intermediate hementin digest using a Mono Q anion-exchange column and the Pharmacia FPLC separation system are depicted. Dhem fragments are eluted between 0.12 and 0.15 M NaCl. Other fragments and fibrinogen (Fg) eluted at higher NaCl concentrations. fragment Yhem i, and fragment Dhem 1 bound mAb 9E9 (second lane). The specific reaction of fragment Dhem 1 with mAb 9E9 along with the calculated molecular weight for its (Y chain remnant strongly suggested that the fragment retained the entire COOH terminus of the fibrinogen LY chain. Furthermore, the data were consistent with late cleavages occurring only in the COOH terminus of the (2 chain. Purification of Fragment Dk,, ,--To isolate fragment D he,,, 1 from an intermediate hementin digest of fibrinogen, the digest was applied to a Mono Q column, and eluted with a linear NaCl gradient (Fig. 6, elution profile). The first protein peak, eluting between 0.10 and 0.15 M NaCl contained mostly Dhem fragments with some contaminating fibrinogen (Fig. 6, SDS-PAGE). The second protein peak, eluting between 0.15 and 0.20 M NaCl, contained mostly fibrinogen, Y hem fragments, and fragment Ehem (Fig. 6, SDS-PAGE). Contaminating fibrinogen was removed from the Dhem pool by affinity chromatography on immobilized anti-E antibodies (data not shown).
Fragment Dhem 1 on separation from other fibrinogen fragments showed high susceptibility to proteolytic degradation. For example, after storage for 1 month at -20 "C, the majority of fragment Dhem 1 had degraded cy chain COOH-terminal peptides. The degradation of (Y chain remnants was prevented by (1) storage at -70 "C, (2) use of sterile technique, and (3) the addition of protease inhibitors (data not shown).
It has long been hypothesized, but not directly proven, that the binding site for plasma fibronectin on fibrinogen is located at the COOH-terminal end of the Aa chain (32). We tested the hypothesis by using affinity chromatography on immobi- A preparation containing a majority of Dhem fragments with degraded oi chains was used to demonstrate the specific binding of Fragment D hem 1 to fibronectin. The initial sample, shown in the first lone was applied in phosphate-buffered saline, pH 7.4. The second lane shows fragments Dhem that did not bind to the column. After washing the column with 1.0 M NaCl, a urea gradient was applied. Fragment Dhem , was eluted with 1.0 M urea and represented the only species bound to the column.
lized plasma fibronectin to separate fragment Dhem I from Dhem fragments with shorter (Y chain remnants. Fig. 7 shows that when a highly degraded pool of Dhem fragments was chromatographed on a column of immobilized fibronectin, all Dhem fragments not possessing the entire (Y chain COOH-terminal region passed through the column (Fig. 7, second lane). The column bound only fragment Dhem i, which was eluted with a 1.0 M urea, 1.0 M NaCl containing buffer (Fig. 7, third lane). The difference in mass between fragment Dhe,,, i and fragment D hem '2, which did not bind, is 12 kDa which corresponds approximately to the COOH-terminal 111 amino acids of the cy chain. The results of affinity purification of fragment Dhem I indicate that the fibronectin binding site is most likely located within amino acids 499 and 610 at the COOH terminus of the Aa chain.
Purified fragment Dheml was used to determine directly the An expanded sequence of the area in the connector region surrounding hementin and plasmin cleavage sites is shown. The specific peptide bonds cleaved by both enzymes are pointed out, illustrating (a) the close proximity of cleavage sites for the two enzymes, (b) the different amino acid specificities exhibited, and (c) the cleavage by hementin of spatially adjacent peptide bonds on different chains, contrasted with plasmin which cleaves nonadjacent peptide bonds.
peptide bonds cleaved in the connector region by hementin. The approach taken was NH*-terminal sequencing of each isolated polypeptide chain remnant derived from the fragment. The covalent bonds between o(, 0, and y chain remnants were broken by disulfide reduction and carboxymethylation. cy, & and y chain remnants were isolated by reverse-phase liquid chromatography on a Cq column. Each chain was subjected to seven or eight cycles on an ABI 430 sequenator (Applied Biosystems, Foster City, CA) with an on-line phenylthiohydantoin analyzer. The resulting sequences were compared with the known amino acid sequence of fibrinogen chains. The NH*-terminal amino acid sequence determined for each chain remnant indicated the hementin cleavage sites in the connector region occurred between Aa Asn'02-Asn'03, BP Lys'""-Gln'31, and y Pro76-Asn77 (Fig. 8).

DISCUSSION
Posterior gland hementin cleaves purified human fibrinogen in a unique manner. The unique cleavage mechanism was reflected in the isolation of fibrinogen fragment Dhem I (Figs. 6 and 7), which retains an intact Acu chain COOH-terminal region (Figs. 4 and 5). Thus, hementin is the only known protease which makes its first cleavages in the connector region of fibrinogen before degradation of the Aa chain COOH-terminal region.
Hementin is also produced by the anterior salivary glands of the leech I-I. ghilianii (25). The pattern of fibrinogen degradation by anterior gland hementin, as assessed by SDS-PAGE (33), seemed similar but not identical to that shown for the posterior gland enzyme. The most significant difference was in the relative rates of cleavage in the Aa chain COOH-terminal region. Anterior gland hementin cleaved in the Aa chain COOH-terminal region much quicker, relative to connector region cleavage, than the posterior gland enzyme. It is for this reason that hementin from posterior salivary glands is more suitable for producing fibrinogen fragments which retain the Aoc chain COOH-terminal domain.
The majority of fibrinogenolytic proteases preferentially cleave in the COOH-terminal region of the Aoc chain. Plasmin (1,2,4,6), trypsin (6, ll), leukocyte (16,18), and platelet (19) proteases, and proteases from many snake venoms (20)(21)(22)(23)(24) all cleave first in the Aa chain COOH-terminal region. The high susceptibility of the Aa chain COOH-terminal region to proteolytic degradation has led to the hypothesis that it is unstructured and surface-oriented (34). However, recent evidence using electron microscopy (35)(36)(37), scanning calorimetry (38), and immunochemical techniques (39) support the concept that parts of the Am chain COOH-terminal region have some ordered secondary structure and are associated noncovalently with the E domain. Our observations revealed increased proteolytic susceptibility of the (Y chain COOHterminal region in fragment Dhem 1 relative to fibrinogen, supporting the idea that the Aa chain in fibrinogen is protected from proteolysis through its interaction with the remainder of the molecule. Besides hementin, the only fibrinogenolytic proteases which do not cleave the (Y chain COOH terminus preferentially are the @-fibrinogenases from Trimeresurus mucrosquamatus (20) and Trimeresurus gramineus (22) snake venoms, and proteases II and III from Crotalus atrox venom (24). These proteases preferentially cleave an NHp-terminal peptide from the BP chain, which contains a polymerization site (7,40). Recent investigation into the structure of the polymerization site on the B/3 chain NH* terminus indicated that it resides on an exposed region (40), consistent with its relative sensitivity to proteolytic degradation. It is interesting that the leech H. ghilianii has evolved to produce an anticoagulant protease which does not cleave first in either the Aa chain COOH-terminal or B/3 chain NHp-terminal regions preferred by other enzymes. The originally intended function of the leech protease involved prevention of blood clotting during feeding. A fibrinogenolytic protease would then be required to act more quickly on its substrate than the blood coagulation cascade. Fibrinogenolytic fragments which lack ALY chain COOH-terminal peptides, such as fragment X, still retain >95% coagulability (3,5). Similarly, the 325-kDa derivative of fibrinogen produced by cleaving the BP chain NH2 terminus with protease III from C. atrox venom is >95% coagulable (41). The most efficient way to degrade fibrinogen into noncoagulable species is to cleave the connector region, splitting the molecule, destroying the bivalent function, and in doing so producing fragments which are themselves anticoagulants (5,9).
Both hementin and plasmin cleave fibrinogen through the three polypeptide chains in the connector region. This was demonstrated by the fact that hementin produced proteolytic fragments of fibrinogen that reacted with anti-D and anti-E antibodies (Fig. 3) in a manner similar to that shown for plasmic fragments Y, D, and E, and the localization of hementin cleavage sites in close proximity to plasmic cleavages sites in the connector region (Fig. 8). Other proteases which presumably cleave fibrinogen in the connector region are trypsin, which produces fragments X, Y, D, and E (6, ll), and leukocyte elastase which produces X-like and D-like fragments (42,43). Unlike hementin, however, both these enzymes cleave first in the Aa chain COOH-terminal region. The cleavage sites for hementin in the connector region were first demonstrated (Fig. l), then proven by sequence analysis (Fig. 8) to exist in close proximity to plasmin cleavage sites. Using the procedure of Chou and Fasman (44) to predict the secondary structure of the area surrounding both hementin and plasmin cleavage sites in the connetor region (results not shown), we derived a region of open structure. The prediction is in agreement with Doolittle and colleagues (45) who used similar methods to predict the structure of the connector region as a coiled-coil, interrupted in the middle by an openstructured, protease-sensitive area. There is little precedent for proteases with specificity for cleaving peptide bonds on the NHz-terminal side of asparagine and glutamine, as demonstrated for hementin. One example is procollagen-N-proteinase that cleaves either Pro-Gln or Ala-Gln peptide bonds to release the procollagen-Npeptides from the collagen triple helix, allowing it to polymerize. Cleavage by procollagen-N-proteinase is also predicted to occur between two regions of triple helix, in an area characterized by lack of interaction between the three procollagen polypeptide chains (46). The significance of the interaction between fibrinogen and tibronectin has long been recognized (47). The affinity purification on immobilized fibronectin of fragment Dhem 1 from other Dhem fragments with shorter (Y chain remnants, provided convincing evidence that the binding site was within the COOH-terminal 12 kDa of the Aa chain. The direct interaction between tibronectin and the Acu chain COOH-terminal domain of fibrinogen reinforces the significance of this domain in the processes of cell migration and wound healing (47).